In the 1970's, a definitive correlation was established between the levels and types of lipoproteins and heart disease. It has been observed that high levels of low density lipoproteins (LDL) can cause an increase in the incidence of heart disease, and that delay in the removal of these particles increases the time they remain in the plasma, exposes them to structural modifications and increases their interaction with the arterial walls. Furthermore, alterations in Apo B reduce the capacity of these particles to bind to their receptors and they are therefore largely recognized by macrophage receptors. The incapacity of the macrophages to regulate the internalization of modified LDL causes an accumulation of cholesterol esters and the formation of foam cells, a phenomenon that favors the development of atherogenesis.
Atherosclerosis is a phenomenon that begins in childhood and adolescence and progresses throughout life. Its consequences, such as arterial occlusion and its will known clinical manifestations (acute myocardial infarction, cerebral vascular accidents, gangrene of the lower limbs, etc.) begin many years before they are detected with the alteration of the vascular walls. Some patients who present serumal lipid levels that are higher than normal, also present a greater incidence of this type of ailment, as is the case of diabetics, nephropaths or congenital hyperlipidemics, among many others. In order to detect the possibility of these patients developing atherosclerosis and its clinical manifestations, the so-called “Coronary Risk Factors” are evaluated. These factors indicate the degree of exposure of the individual to circumstances that may determine a greater risk of presenting ailments related to this possibility; that is, they are used to determine the risk of atherogenesis. Coronary risk factors are as follows:                High low density total cholesterol and lipoproteins        High blood pressure        Smoking        Diagnosis of ischemic cardiopathy        Hypoalphalipoproteinemia (Low levels of high density lipoproteins or HDL)        Diabetes        Obesity        Family history of premature heart disease        Masculine sex        Proteinuria        Hypertriglyceridemia        
In order to establish if the patient is exposed to coronary disease risk factors, directed interrogations, exploration, determination of the lipid profile, electrocardiogram and radiograph of the thorax are performed. When peripheral vascular insufficiency is suspected, a Doppler Ultrasound and arteriography are included.
All the elements considered in this evaluation are used in the detection and follow-up of patients who are within the so-called “risk groups”, which include individuals, who due to different pathologies, show one, some are all of the coronary risk factors. However, there is a large number of persons at risk from atherogenesis who have not been detected as they do not yet present the related clinical manifestations and therefore have not been placed within the risk groups. As there is no early diagnosis of these individuals, many valuable years of prevention are lost and when clinical manifestations do present themselves, the damage is mainly irreversible.
Furthermore, studies that assess atherogenesis risk factors based on the concentration of lipoproteins in the plasma of subjects included in some of the risk groups are inconsistent. Then, there are patients who are included in the risk groups who do not present the typical clinical description that denotes risk of atherogenesis, such as painless development with only a sensation of fatigue and lack of air, and some others go by unnoticed as they are confused with various pathologies. Hence, it is necessary to broaden the examination of the patient in the search for atypical manifestations.
There are also cases in which even when individuals are exposed to risk factors they do not develop atherosclerosis. Not all the factors to be assessed to determine the risk of atherogenesis are reliable. Some of the most widely discussed ones are included in the lipid profile, such as the high levels of total cholesterol and LDL and low levels of HDL in the blood, which are used to calculate the atherogenic index. This index is an arbitrary parameter that has experimentally proved to be unreliable in evaluating risk of atherogenesis: For example, in tests with rabbits submitted to diets high in cholesterol for long periods of time, high levels of total cholesterol, free cholesterol, esterified cholesterol and cholesterol associated with LDL, low levels of HDL associated cholesterol, hypertriglyceridemia, a high percentage of esterification and a high atherogenic index were obtained; however, they did not present atherogenesis.
In conclusion, the method for evaluating risk of atherogenesis used at present has severe limitations; first, the size and type of population likely to be evaluated is limited, due to the cost in time and money that is implied in carrying out the diagnosis tests and this makes the early detection of individuals at risk of atherogenesis who are not placed in risk groups difficult, therefore prevention of its complications is deficient. Second, even when the diagnosis parameters used at present allow certain certainty, most of them are qualitative and some of the quantitative parameters, the most important ones, are under discussion and it is therefore only possible to speak of a “high clinical suspicion of risk of atherosclerosis” since the results are not completely reliable. Third, not much is known about the homeostasis of lipids in humans and in mammals in general and it has not been possible to establish a clear relationship between many of the parameters considered as atherogenesis risk factors and their clinical manifestations, hence the determination of said risk cannot lie on solid bases, without a true understanding of the factors intervening in atherogenesis and their clinical manifestations.
There are other factors that can be useful in establishing the risk of atherogenesis in a more reliable way, such as the determination of the levels of the cholesterol ester transferring protein (CETP) in the plasma. This protein has been widely studied and is one of the best known factors intervening in lipid homeostasis. CETP has an important role in lipoprotein metabolism and in the development of arterial coronary disease, since it tends to generate high levels of LDL and VLDL that are associated with the progression of atherosclerosis (Marotti-K R, Castle-C K, Boyle-T P, Lin-A H, Murray-R W and Melchior-G W, 1993, Nature 364(6432):73-5). CETP is a multifunctional protein that promotes the exchange of cholesterol esters between HDL and LDL and the exchange of cholesterol esters and triacylglycerols between HDL and VLDL; its effect on the catabolism of HDL has an influence on its cholesterol ester content as well as on its composition, size and spherical structure (Rye-K A, Hime-N J & Barter-P J, 1995, J. Biol. Chem. 270(1):189-196) (Bruce-C, Chouinerd-R A y Tall-A R, 1998, Annu. Rev. Nutr. 18:297-330).
CETP also participates in the recycling of cholesterol deposited in the peripheral tissues during lipolysis of the lipoproteins (Jiang-XC, Moulin-P, Quinet-E, Goldberg-I J, Yacoub-L K, Agellon-L B, Compton-D, Schnitzer-Polokoff-R and Tall-A R, 1991, J. Biol. Chem. 266(7):4631-9) (Nagashima-M, McLean-J and Lawn-R, 1988, J. Lipid. Res. 29:1643-1649) (Tall-A, 1995, Annu. Rev. Biochem. 64:235-257). This transport, which we call “cholesterol reverse transport” confers on CETP the character of an anti-atherogenic protein, hence its importance in susceptibility or resistance to atherosclerosis (Kondo-I, Berg-K, Drayna-D, Lawn-R, 1989, Clin. Genet. 35(1): 49-56) (Bruce-C, Chouinerd-R A y Tall-A R, 1998, Annu. Rev. Nutr. 18:297-330). In accordance with the above, the factor that determines its anti-atherogenic capacity is not the level of HDL in plasma, but the distribution of sizes of its population, which has a strong correlation with CETP levels (Brown-M L, Inazu-A, Hesler-C B, Agellon-L B, Mann-C, Whitlock-M E, Marcel-Y L, Milne-R W, Koizumi-J, Mabuchi-H, 1989, Nature 342(6248):448-51).
In experimental work, it has been observed that mammal species lacking CETP in a normal way are resistant to developing arterial heart disease.
In contrast, in transgenic individuals of these same species that express CETP a decrease in size and HDL levels can be observed. As a consequence, CETP expression increases susceptibility to suffer from diet induced arterial heart disease (Rye-KA, Hime-NJ & Barter-PJ, 1995, J. Biol. Chem. 270(1):189-196). Similarly, it has been observed that humans with a genetic deficiency of CETP present HDL levels that are much higher than the levels of normal subjects (hypoalphalipoproteinemia). These individuals seem to have a lower incidence of heart disease (Inazu-A, Quinet-E M, Wang-S, Browun-M L, Stevenson-S, Barr-M L, Moulin-P and Tall-A R, 1992, Biochemistry 31(8):2352-8). However, transgenic mice with hypertriglyceridemia that express CETP are protected against atherogenesis (Homanics-G E, de Silva-H V, Osada-J, Zhang-S H, Wong-H, Borensztajin-J and Maeda-N, 1995, J. Biol. Chem. 269:16376-16382).
This invention is related to laboratory tests designed to identify and quantify the cholesterol ester transferring protein (CETP) in both biological and synthetic samples for clinical use, with the purpose of evaluating risk of atherogenesis and for use in research related to CETP.